1. Field of the Invention
The present invention relates to an analytical element for dry analysis, and more particularly to an analytical element appropriately employable for dry analysis operation of substance contained in a body fluid in micro-quantity.
2. Description of Prior Arts
As a method of analysis of substance contained in a liquid sample such as a body fluid in a micro-quantity, there has been conventionally utilized a wet analysis such as a method comprising steps of bringing said substance into contact with other substance contained in a solvent so as to undergo a detectable reaction such as a reaction directly or indirectly producing or changing color, and detecting said reaction.
However, a dry analysis (i.e., dry analysis operation) developed for the purpose of simplifying the analytical procedures has been recently utilized. As a representative example of the analytical element utilized for the dry analysis, well known is a dry analytical element (also called a dry analytical material or article) which is in the form of a sheet, film, strip or tape basically comprising at least one reagent layer containing a reagent which directly or indirectly undergoes detectable reaction in contact with the substance under analysis (analyte), and a support thereof. The analysis employing the analytical element is generally carried out by procedures of applying a liquid sample containing the analyte onto the analytical element, subjecting the analytical element to incubation if necessary, measuring thus produced detectable reaction such as generation or change of color by means of a photometry or the like, and determining the amount of analyte according to colorimetry.
There are known a variety of analytical systems utilized in analysis of analyte employing the analytical element. Representative examples of the analytical system are as follows.
(A) An analytical system for detecting and measuring a detectable reaction between a reagent contained in a reagent layer and an analyte, such as generation or change of color. This analytical system is employed for analyses of analytes such as a variety of proteins, for instance, total protein, albumin and globulin, hemoglobin decomposition substances, for instance, free (non-conjugated or indirect) bilirubin and conjugated (direct) bilirubin.
(B) An analytical system employing a reagent layer containing at least two kinds of reagents, which comprises procedures of reacting one of the reagent with the analyte to produce a reactive substance such as ammonia or hydrogen peroxide, subsequently reacting the reactive substance with other reagent (e.g. dye precursor) in contact therebetween to cause a detectable reaction such as generation or change of color, and detecting and measuring the reaction. This analytical system is employable for analysis where analyte is glucose; lipids such as cholesterol, triglyceride and free fatty acid; enzymes such as lactate dehydrogenase; urea and uric acid.
(C) An analytical system employing a reagent layer containing a non-diffusive reagent having a color-forming group, which comprises procedures of converting the reagent into a diffusive product carrying the color-forming group by reaction with analyte, separating the diffusive product from the unreacted non-diffusive reagent, causing a detectable reaction such as generation or change of color in contact between the diffusive product and a chromogen substance such as coupler, and detecting and measuring the reaction. This analytical system is employable for analysis of polysaccharide hydrolase such as amylase.
(D) An analytical system employing a reagent layer containing a non-diffusive reagent having a detectable property (e.g., color), which comprises procedures of producing a diffusive product carrying the detectable property from the reagent by reaction with analyte, separating the diffusive product from the unreacted non-diffusive reagent, and detecting and measuring the diffusive product. This analytical system is employable for analysis of polysaccharide hydrolase such as amylase.
As described above, the analytical element contains a reagent reactive to analyte and may further contain another reagent reactive to a product produced by the reaction between the analyte and the former reagent. These reagents are selected appropriately to meet the purposes of the analysis. Among these reagents, not a few kind of reagents easily deteriorate under unsuitable surrounding pH conditions, or require specific pH conditions to show their reactivities. Accordingly, these reagents should be used under consideration on adjustment of their surrounding pH conditions.
For instance, a number of analytical detection systems using the dry analytical elements utilize enzymic reactions, in which the enzyme is an analyte, or is utilized as a reagent to decompose the analyte. The enzymic reaction generally proceeds smoothly in a pH range specifically adjusted to meet the requirement of the utilized enzyme. For this reason, the conventional dry analytical element is prepared to provide an optimum pH value for the enzyme to be utilized to the area (surroundings) where the predetermined enzymic reaction takes place. More in detail, the analytical element utilizing an enzyme as analyte contains a buffer reagent (known as a buffer reagent in the wet analysis) within a layer where the enzymic reaction is to take place, for instance, a layer containing a substrate of the enzyme. Otherwise, the analytical element utilizing an enzyme as a reagent for decomposing an analyte contains the buffer reagent within, for instance, a layer containing the enzyme. Thus, the conventional analytical element is adjusted on the surrounding pH conditions so as to allow smooth progress of the enzymic reaction.
In the analytical detection system, a detection system where the enzymic reaction product per se shows a color formation indicative of the amount of the analyte is known. Also generally known is a detection system where a reactive product produced by an enzymic reaction such as hydrogen peroxide reacts with a color-forming reagent (e.g., dye precursor) contained in the element to show color formation or color change. As the dye precursor, an azo-dye precursor such as a diazonium salt is known. The diazonium salt, however, has a problem that it is poor in preservability under the optimum surrounding pH conditions of most enzymes (pH range most preferable for the enzymic reaction). This means that: if the surrounding pH condition within the analytical element is set to meet the conditions appropriate for the enzymic reaction, the diazonium salt contained in the element decomposes or deteriorates in the course of storage of the element, while if the surrounding pH condition is set to meet the condition appropriate for preservation of the diazonium salt, the enzymic reaction utilized for the detection hardly proceeds smoothly.
For complying with the above-mentioned problem, it can be proposed that a diazonium-containing layer and a layer where the enzymic reaction is performed be independently arranged in the analytical element to incorporate a buffer reagent into only the latter enzymic reaction layer. However, even in this arrangement, a low molecuar weight buffer reagent permeates and diffuses from the enzymic reaction layer into the former diazonium-containing layer in the course of storage of the element. Accordingly, the decomposition or deterioration of the diazonium salt still occurs. The former diazonium-containing layer and the latter enzymic reaction layer should be brought into fluid contact (i.e., contact via fluid) with each other when the analytical operation is carried out. For this reason, these two layers cannot be arranged under complete separation.
A further problem is as follows. A reagent layer of a conventional analytical element utilizing an oxidase is prepared using a coating solution containing oxidase and a reduction-type leuco dye. In this example, the problem is: if the pH condition of the coating solution is adjusted to correspond to the optimum pH condition of the oxidase, the leuco dye is partially oxidized through oxidation reaction caused by oxygen in air in the course of preparation or storage of the analytical element. Accordingly, the leuco dye is liable to show color prior to performing analysis, resulting in reduction of the analytical accuracy.
As described above, the conventional analytical element hardly gives satisfactorily accurate results, as far as such detection system that there is observed difference between the optimum pH range for preservation of one or more reagent contained in the element and the optimum pH range for performing the detection reaction in the analytical operation is concerned. This problem is hardly solved, so far as the conventional buffer reagent is employed. For the same reason, two or more reagents having optimum pH ranges for preservation not being overlapped with each other are hardly contained in one analytical element.